AbstractOcean acidification influences photosynthesis, respiration, and metabolism in marine diatoms, leading to changes in diatom growth performance and shifts in phytoplankton communities. Previous studies have demonstrated that increases in seawater CO₂ concentrations affect the uptake of trace metals such as iron, zinc, copper, and cobalt by marine diatoms. However, the influence of increased CO₂ on calcium, which plays a vital role as a secondary messenger in various signaling pathways within organisms, has received limited attention so far. This study examined the effect of increased CO₂ on Ca homeostasis and signaling in the marine diatom Phaeodactylum tricornutum. While seawater acidification had little effect on the diatom's growth, it significantly changed cell properties (surface topography, adhesion, and surface potential). Elevated CO₂ concentrations reduced calcium accumulation P. tricornutum and lowered the rise of cytosolic Ca²⁺ transients stimulated by toxic aldehyde, phosphorus supplement, and hypo‐osmotic stress. Our results suggest that a continuous rise in atmospheric CO₂ may alter diatoms' response to environmental cues.